Polypeptides are commonly analyzed using ligand binding assays (LBA) such as enzyme-linked immunosorbent assays (ELISA). However, ELISA can lack specificity and accuracy, for example due to cross reactivity and an inability to distinguish similar molecules such as analogs and metabolites. Use of ligand binding assays such as ELISA for analyzing polypeptides is also hindered when suitable antibodies are not yet available for the polypeptide of interest. Development of antibodies can be an expensive and time-consuming process.
Although biologics have historically been quantified using ligand binding assays (LBAs), over the past few years, there has been a trend toward the analysis of large molecules by liquid chromatography tandem mass spectroscopy (LC-MS/MS). However, intact polypeptides such as insulin are particularly difficult to analyze by LC-MS/MS. For example, mass spectroscopy (MS) sensitivity can be low due to poor transfer into the gas phase and poor fragmentation due to the presence of multiple stabilizing disulfide bonds. In addition, polypeptides can suffer from non-specific binding and poor solubility, making liquid chromatography (LC) and sample preparation method development difficult.